PROJECT SUMMARY/ABSTRACT The trophectoderm layer of the blastocyst stage embryo is the precursor for all trophoblast (TB) cell types in the placenta ? the villous cytotrophoblasts (vCTBs), syncytiotrophoblast (STB), and extravillous trophoblasts (EVTs) comprising column cytotrophoblasts (cCTBs) and invasive cytotrophoblasts (iCTBs). Abnormalities in TB development result in placental pathology, and are associated with adverse outcomes during pregnancy. Yet, despite being critically important to maternal and fetal health, human TB development remains poorly understood. Mechanistic studies on human TB development are difficult due to challenges in research with human embryos, and limited availability of placental samples from early gestation. Further, human placental samples are associated with heterogeneity, containing multiple TB and non-TB cell types, and have likely undergone adaptation to the maternal environment. To overcome these limitations, we propose to use TB derived from human embryonic stem cells (hESCs) as a model system for studies on human TB development. However, the molecular mechanisms underlying the acquisition of TB fate in hESCs remain unclear. This lack of understanding is a major impediment for widespread use of hESC-derived TB as a model system for early human TB development. A major challenge in this context is the lack of defined culture conditions for initiating TB differentiation of hESCs and/or differentiation of hESC-derived TB to specialized TB subtypes. Therefore, here we propose to validate a completely defined culture system for TB differentiation of hESCs, including selective differentiation to STB or iCTBs. Accordingly, in Aim 1, we will validate a culture system based on the completely defined E6/7/8 medium, and vitronectin-coated tissue culture plates. Specifically, we will investigate the expression of a panel of biomarkers to confirm that bona fide TB sub-types can be obtained using these conditions. In Aim 2, we will investigate whether TB differentiation of hESCs to STB and iCTBs can occur in defined conditions where exogenous bone morphogenetic protein (BMP) is not added, or where BMP signaling through the SMAD1/5/8 signaling pathway is inhibited. Finally, in Aim 3, we will test the hypothesis that Rho/ROCK signaling downstream of S1P activation mediates TB differentiation of hESCs in the proposed culture system. The significance of our work relates to its relevance to our understanding of early human TB development. Validation of defined culture conditions for TB differentiation of hESCs will enable mechanistic studies on TB differentiation of hESCs, and differentiation of hESC-derived TB to specialized TB sub-types. This in turn will enable routine acceptance use of hESC-derived TB a model system for early TB development. Such a model system will be a powerful tool for advancing understanding of early human placental development.